Production of neural protective and regenerative factors from stem cells and treatment of nervous system conditions therewith

ABSTRACT

A method for producing stem cell conditioned media for treatment of neurological insults is provided herein. By providing for the culture of adipose stem cells, and collecting the supernatants thereof, the supernatants have been shown to effect biological activity in preventing neural death when the supernatants are administered to a patient that has or is about to be subjected to a neural insult.

CLAIM OF PRIORITY

The present application claims priority to U.S. Provisional PatentApplication No. 60/823,460, filed Aug. 24, 2006, which is incorporatedby reference herein.

BACKGROUND

Pluripotent cells, sometimes referred to as stem cells, arecharacterized by an ability to differentiate into a variety of differentcells. Some pluripotent cells types, such as human embryonic stem cells,display an ability to differentiate into the broadest spectrum of cells;in fact, embryonic stem cells display an ability to differentiate intopractically any type of cell that exists within the human tissues.However, as embryonic stem cells develop and differentiate into lines ofpartially and/or fully differentiated cells, those furtherdifferentiated cells lose some or all of their pluripotent abilitybecause embryonic stem cells have the ability to “morph” intopractically any cell type, the scientific community has explored thepossibility of using these embryonic stem cells to replace those injuredor dying cells in individuals suffering neurodegenerative disease suchas parkinson's disease.

However, embryonic stem cells have limitations in their ability to beused clinically, as they must be derived from another individual—anembryo. This not only raises a potential that the patient will rejectthe cells, but it also severely limits the ability for such cells to beused in the first place. Therefore, much effort has been made in findingpluripotent cells that are obtainable in large quantities, that candifferentiate into a target cell, and that will not be rejected by theindividual being treated thereby. One such pluripotent cell that hasbeen used for autologous cell therapy to regenerate neural tissue is thepluripotent cells found in the “stromal” or “non-adipocyte” fraction ofthe adipose tissue. These pluripotent cells were previously consideredto be pre-adipocytes, i.e. adipocyte progenitor cells (hereinafter“adipose stem cells” or “ASC”). Zuk, 2001. Data suggests that theseadipose stem cells have a wide differentiation potential, as research byZuk using subcutaneous human ASCs in vitro were able to bedifferentiated into adipocytes, chondrocytes and myocytes. Id. Furtherstudies by Erickson et al., showed that human ASCs could differentiatein vivo into chondrocytes following transplantation intoimmune-deficient mice, and studies by Stafford showed that human ASCswere able to differentiate into neuronal cells. (Erickson, 2002);(Stafford, 2002). More recently, it was demonstrated that human ASCswere able to differentiate into neuronal cells, osteoblasts (Dragoo,2003), cardiomyocyte (Rangappa, 2003; Planat-Benard, 2004), andendothelial cells (Planat-Benard, 2004). As such studies suggest thatthe delivery of certain pluripotent cells to neural tissue damaged bystroke or cardiovascular disease may cause regeneration of the damagedtissue through differentiation of the delivered pluripotent cells.

Therefore, treatments using autologous pluripotent cells, and ASCs inparticular, have necessarily centered upon the harvest and concentrationof the pluripotent cells from a remote area of the patient to betreated, followed by application of those concentrated pluripotent cellsto an injured or targeted site so that the pluripotent cells candifferentiate and take the place of the damaged cells at the target.See, e.g., U.S. Pat. No. 7,078,230 to Wilkison et al.; U.S. Patent App.Pub. No. U.S. 2005/0260174 to Fraser et al.

SUMMARY

At least embodiment of the present disclosure provides harvestingadipose stem cells (ASC) recovered from adipose tissue to provide anautologous source of cells. These pluripotent cells, which reside in the“stromal” or “non-adipocyte” fraction of the adipose tissue, have thecapacity to differentiate in culture into adipocytes, chondrocytes,osteoblasts, neuronal cells, and myotubes. ASCs can be obtained in largequantities, in the range of 10⁸ to 10⁹ cells, following routineliposuction of subcutaneous adipose tissue. The ready accessibility ofthese cells provides for a particularly feasible and attractive form ofcells for harvest and presents the opportunity to retrieve a givenpatient's own cells as a source of pluripotent cells for harvesting.

Another embodiment of the present disclosure relates to the use ofharvested ASCs or other stem cells to secrete bioactive levels oftherapeutic proteins that can promote repair of injured and diseasedneural tissues or prevent neural tissue death under circumstances thatwould ordinarily result in apoptosis. According to one aspect, stillanother embodiment relates to using media exposed to ASCs maintainedand/or growing in cell culture to produce a composition that promote thegrowth, health, protection, and/or development of various types of humanand animal cells, especially neural cells.

Yet another embodiment of the present disclosure relates to the use ofat least one factor produced by ASCs to effect changes in other cellsexposed to the factors. In one embodiment, at least one factor producedby an ASC is used to prevent the death of neuronal cells either inculture or in the central or peripheral nervous system of an adult ordeveloping animal, including Homo Sapiens.

Still another embodiment of the present disclosure related to a methodof treating diseases, disorders or injuries in neural tissue by exposingneuronal tissues and various cells therein to products produced by ASCs.

Still another embodiment of the present disclosure relates to usingmedia exposed to ASCs to modulate physiological processes such asformation of new vessels or expansion of existing vessels within thecentral nervous system, peripheral nervous system, or spinal cord. Atleast one embodiment relates to a method of treating at least a portionof the central nervous system comprising the steps of administering atleast a fractionated portion of the media exposed to ASC to at least oneregion of the Central Nervous System in either a human or animalpatient.

At least one embodiment of the present disclosure relates to a method ofusing substances secreted by ASCs into the cell culture medium tomodulate at least one of several in vitro neuronal injury pathways.

Still another embodiment of the present disclosure relates to recoveryof at least one compound produced by ASCs in cell culture to a cellculture media in contact with ASCs to modulate the activity of neuronalcells.

Yet another embodiment of the present disclosure relates to using atleast substance derived from ASC media cultured in vitro to treat cellsin vivo at risk for ischemia-hypoxia-induced neuronal death.

Another embodiment of the present disclosure relates to the in vitro useat least one of substances derived from ASC cells cultured in vitro toaffect the activity, viability and/or differentiation of neuronal cellseither in vivo or in vitro.

Still another embodiment of the present disclosure is a method oftreating hippocampal tissue that has been damaged by neonatalhypoxia-ischemia comprising the steps of providing at least substance orcompound derived from media recovered from cultures of ASCs andadministering at least one dosage of the at least one factor tohippocampal neuron tissue and/or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Phase contrast micrographs of (A) human, (B) mouse, and (C) ratgrown in EGM2MV.

FIG. 2: In vitro expansion of (A) human and (B) mouse ASCs.

FIG. 3: Profile of cytokines expressed by human ASCs cultured undernormoxia (A) or hypoxia (B). Conditioned media was applied to a RayBioantibody array VII and developed for visualization. Numbers refer toantibodies (in duplicate) for specific proteins. A “+” or “−” signsignifies positive or negative controls, respectively. The proteins(with detection limits in pg/ml) are: 1, angiogenin (10); 2, MCP-1 (3);3, IL-1ralpha (10); 4, IL-5 (1); 5, IL-6 (1); 6, MIP-3a (100); 7, SCF(10); and 8, TNF-beta (1000).

FIG. 4: Influence of ASC-Conditioned Medium on endothelial cell ecSurvival and prevention of apoptosis.

FIG. 5: Conditioned media from ASCs (stromal cell-conditioned medium:ASC Conditioned Media) protects CGN neurons against potassium (5mM)-induced apoptosis. The rat ASCs were cultured in EGM2MV media toconfluence, and then switched into basal media Eagle (BME with 5 mM K⁺,Invitrogen) for 24 hours. The conditioned media (ASC Conditioned Media)were collected from the ASCs culture and subsequently added to theregular BME (5 mM K⁺) at volumes equivalent to 30% of the total mediavolume. The BME with ASC Conditioned Media was then added to rat CGNcultures. Viable neurons was quantified by counting fluorescein (green)positive cells which result from the de-esterification of fluoresceindiacetate (FDA, Sigma, 10 μg/ml, 5 min) by living cells. Propidiumiodide (PI, Sigma, 5 μg/m, 5 min), which interacts with nuclear DNA ofdead cells, producing a red fluorescence, was used to identify deadneurons (Du, 1997a). A) untreated CGN (control). B) cultures exposed toLK (5 mM) BME for 24 h. C) cultures exposed to LK (5 mM) media with 30%ASC Conditioned Media for 24 h. Data are from a representativeexperiment repeated twice with similar results.

FIG. 6: ASC Conditioned Media protects CGN neurons against LK-inducedapoptosis in a dose-dependent fashion. The rat ASC Conditioned Media(2-30% of final vol) was added to the rat CGN cultures following LKtreatment. The cultures were then double stained with FDA and PI (asdemonstrated in FIG. 5). Data are from a representative experimentrepeated twice with similar results.

FIG. 7: ASC Conditioned Media protects CGN neurons against glutamate (50μM)-induced neuronal death. The ASC Conditioned Media was collected andsubsequently added into the CGN cultures (30% or 50% replacements) thatwere then challenged by 50 μM of glutamate. Neuronal viability wasquantified by staining neurons with FDA (Du, 1997). G indicates that theCGN were exposed to 50 μM glutamate; SCM+G, CGN exposed to 50 μMglutamate and the indicated percentages of ASC Conditioned Media.

FIG. 8: ASC Conditioned Media collected from fresh (P0) or passage 3(P3) ASCs protects CGN neurons against glutamate (50 μM)-inducedneuronal death differently. The 50% replacement of P0 ASC ConditionedMedia exerts a stronger neuroprotective effect than P3 ASC ConditionedMedia. Neuronal viability was quantified by staining neurons with FDA(Du, 1997). Control is CGN without 50 μM glutamate exposure; G indicatesthat the CGN were exposed to 50 μM glutamate; SCMP0+G and SCMP3+G, CGNexposed to 50 μM glutamate and 50% ASC Conditioned Media from cells atP0 or P3, respectively.

FIG. 9: ASC Conditioned Media collected from fresh ASCs has beenenriched 50× using 10K CentriPlus protects CGN neurons against glutamate(50 μM)-induced neuronal death. Addition of 250× enriched ASCConditioned Media almost completely protects neurons against glutamatetoxicity in CGN. Neuronal viability was quantified by staining neuronswith FDA (Du, 1997). G indicates that the CGN were exposed to 50 μMglutamate; G+CSCM CGN exposed to 50 μM glutamate and the indicatedpercentages of 250× Concentrated ASC Conditioned Media.

FIG. 10: Glutamate treatments induced JNK and p38 phosphorylation inCGN. Immunoblot analyses were performed with antibodies againstphosphorylated JNK and p38 (p-JNK and pp 38), and p38 (Santa Cruz).Glutamate treatments increase p-JNK and pp 38 by 3 h post-treatment.Note that glutamate treatment fails to alter total p38 expression in thesame samples. C=control (no glutamate treatment). Glu=glutamatetreatment. Each condition represents 3 samples.

FIG. 11: ASC Conditioned Media collected from P3 human ASCs (HASCConditioned MediaP3) protects CGN neurons against glutamate (50μM)-induced neuronal death. The 50% replacement of P3 HSMC exerts astrong neuroprotective effect on rat CGN followed by glutamatetreatments. Neuronal viability was quantified by staining neurons withFDA (Du, 1997). Control, CGN without 50 μM treatment; G, CGN exposed to50 μM glutamate; 50% HSCMP3+G, CGN exposed 50 μM glutamate and 50% ASCConditioned Media.

FIG. 12: ASC Conditioned Media protects CGN neurons against H₂O₂ (50μM)-induced neuronal death. The ASC Conditioned Media was collected andsubsequently added into the CGN cultures (at ⅓ of the final volume) thatwere then challenged by adding 50 μM of H₂O₂. Neuronal viability wasquantified by staining neurons with MTT (Du, 2003). C, untreated CGN;H202 50 μM, CGN exposed to 50 μM H₂O₂ H202+SCM 30%, CGN exposed to 50 μMH₂O₂ and 30% ASC Conditioned Media; STM 30%, CGN exposed to 30% ASCConditioned Media only.

FIG. 13: ASC Conditioned Media treatment improved cortical neuronsurvival following oxygen and glucose deprivation (OGD) injury. ASCConditioned Media (added to a final concentration of 5%-100% byreplacing the medium) protects cortical neurons in a dose-dependentfashion. SCM, ASC Conditioned Media added at the indicated percentages.

FIG. 14: ASC Conditioned Media prevents neuronal loss when administeredto neonatal rats at 24 hours following hypoxic-ischemic injury.Representative coronal sections of postnatal day 14 (P14) rat brainsdemonstrate that 7 days following unilateral (left) carotid ligation andexposure to hypoxia (7%) for 2 hours at P7 (Wei, 2004) with or withouttreatment with ASC Conditioned Media (10 μl/pup, 24 h following H-Itreatments). a. normal, b. ischemia-treated with BME, c. ischemiatreated following 24 h post treatment with ASC Conditioned Media. Notethe moderate damage in the hippocampus ipsilateral to carotid ligationin animals (compare a vs. b) and the significant protection by ASCConditioned Media (b vs. c). Rectangle indicates the lesion site in theleft hippocampus. The area of tissue in the hippocampus ipsilateral tothe lesioned hemisphere was compared in the same animal with the area oftissue remaining in the matching brain region contralateral tounlesioned hemisphere. The percentage area loss was then determined ineach animal, and data are presented as the mean plus or minus SEM foreach group. ASC Conditioned Media 24 hours after hypoxia “significantly”protects against hippocampal volume loss induced by ischemic injury(73±3 vs. 90±1, n=2/group, one-way ANOVA, * p<0.05).

FIG. 15: Results of proteomic analysis of ASC Conditioned Mediarecovered from media contacted with ASCs in vitro. Specific proteinsidentified in this experiment include, but are not limited to, proteinsrelated to neuroprotection: NGF (nerve growth factor), GDNF(glia-derived neurotrophic factor, IGF-1 (Insulin-like growth factor,and VEGF (vascular endothelia growth factor).

FIG. 16: As expected, in inactivation of a single factor present in ASCConditioned Media, which is known to limit damage due to a specificinsult, significantly reduces the ability of ASC Conditioned Media toprotect against the specific damaging agent. Injury was induced in CGNby exposure to 40 μM glutamate as described above. ASC ConditionedMedia, protected the CGN from damage (G+ASC). Pretreatment of ASCConditioned Media with an anti-BDNF antibody that neutralizes BDNFactivity significantly, but not totally, reduced the ability of ASCConditioned Media to protect CGN (G+antiBDNF+ASC). Conversely, it wouldnot be expected that BDNF activity would protect neurtrons from injurydue to other damaging agents or conditions, such as OGD, oxidation(e.g., H₂O₂), or toxins (E.g., MPP).

FIG. 17 FIGS. 17 a and 17 b: Results of tests showing that ASC-CMpreserves the cognitive function of rats following hypoxia-ischemiainjury, and utilizing the Morris Water Maze test.

FIG. 18 displays the results of an experiment indicating that ASC-CMprotects neurons against 6-hydroxydopamine (6-OHDA)-mediated death.

FIG. 19 displays the results of PC12 cells were cultured in DMEMcontaining 10% FBS for 3 days, then starved in BME medium without FBSfor 24 hours. Various percentages of the medium was exchanged for anequivalent volume of ASC-CM, as indicated in FIG. 19.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thedisclosure, reference will now be made to the embodiments illustratedherein and specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended.

As used herein, a therapeutically effective dosage or amount of acompound is an amount sufficient to affect a positive effect on a givenmedical condition. The affect, if not immediately, may, over period oftime, provide a noticeable or measurable effect on a patient's healthand well being.

According to one aspect of the present disclosure, it has been foundthat when ASCs are cultured in vitro, the ASCs secrete a combination ofangiogenic and antiapoptotic factors and/or additional compounds (eitheras single factors, or in combination with one another) in relativeconcentrations and combinations that have been shown to exert effectiveneuroprotection in a variety of mechanistically distinct neuronal deathpathways. As a result, according to one embodiment, a therapeuticallyeffective dose of ASC secretions from in vitro culture is administeredto prevent or counteract a chronic or acute neural injury or insult.

In particular, according to at least one embodiment, it was found thatmedia used to culture and/or maintain ASCs in vitro has the unexpectedcharacteristic of protecting neural tissue and/or encouragingregeneration from stimuli-induced damage when administered to a patientin a therapeutically effective dosage. This media, or extracts thereof,appears to inhibit critical neuronal death pathways due to the presenceof several complementary neuroprotective factors which combine to limitneuronal death and/or stimulate regeneration of neural cells in vitro aswell as in vivo in the context of neural insult, although the mechanismis not entirely understood. Further, fractions of media conditioned byASCs are a source of various factors that either alone or in combinationwith one another have shown an ability to affect neuronal cells that aresubject to acute or chronic injury. Accordingly, the ASC conditionedmedia has been shown to be an excellent source of material useful forproducing, concentrating, and isolating a broad spectrum combination ofcompounds in relative percentages and forms that effect a therapeuticeffect when administered to an individual suffering from a neuralinsult, including central nervous system or peripheral nervous systeminjuries that may be chronic or acute in nature. Further, ASCconditioned media has been shown to be an excellent source of materialuseful for producing, concentrating, and isolating individual compounds,or groups of compounds, that have been shown to protect neuronal cellsfrom death, damage and insult and/or to cause regeneration thereof.

For example, the ASC conditioned media and/or fractions andconcentrations thereof have proven effective to treat or prevent variousdisorders that involve hypoxia-ischemia (H-I) of the brain includingneonatal or adult H-I-induced encephalopathy, stroke andneurodegenerative disorders, and such treatments do not carry the samerisk of rejection as that shown by injecting foreign stem cells into apatient, since at least one embodiment does not inject any cellswhatsoever into a patient. Further, application of ASC conditioned mediaand/or fractions and concentrations thereof can be used to treat chronicor acute injuries in the peripheral nervous system, central nervoussystem, and/or spinal cord in either neonatal individuals, children, oradults.

It will be appreciated that other stem cells or pluripotent cells may beutilized, such as other mesenchymal stem cell (MSC), which are found inthe stroma of different tissues throughout the body. Human MSCs(including ASCs) are characterized by the surface marker profile oflin−/CD45−/c-kit−/CD90+. Further, in one embodiment, appropriate stemcells display the CD34+ positive at the time of isolation, but lose thismarker during culturing. Therefore the full marker profile for one stemcell type that may be used according to the present application islin−/CD45−/c-kit−/CD90+/CD34. In another embodiment utilizing mouse stemcells, the stem cells are characterized by the Sca-1 marker, instead ofCD34, to define what appears to be a homologue to the human cellsdescribed above, with the remaining markers remaining the same. It willbe appreciated that other stem cells with similar marker profiles couldbe used, such as the pluripotent stem cell from skeletal muscle that wasidentified by Case et al (Annals of NY Acad Sci. vol. 1044:183-200).Case indicates that these cells appear to exist in the adipose and braintheorize that these are the same stem cell that resides in manydifferent organs. They have termed these cells common pluripotent stemcells (CoPSC), but other stem cells could be used, with the exception ofhematopoietic stem cells. This would also include progenitor cells thatderive from MSCs.

The process for producing the ASC conditioned media is further outlinedbelow, as is the method for using the media or its fractions ordistillates to treat nervous system insults or neurodegenerativeconditions. While the term “ASC conditioned media” is used throughout,it is understood that the same processes will apply to production ofsimilar media through other stem cells, but that adipose stem cells areused for exemplary purposes.

I. Process for Producing ASC-Conditioned Media

A. Isolation and Expansion of Adipose Stem Cells

To isolate human ASCs, lipoaspirates (250-500 ml) were obtained frompatients undergoing elective liposuction procedures and processedessentially according to Zuk et al. ASCs were plated at 1,000-10,000cells/cm². Most ASCs attach to the flask and, when cultured in EGM2MVmedia for example, can be multiplied 50-fold in 8 days (FIGS. 1 and 2)with their growth rate decreasing when they reach confluency. FIG. 2Bdemonstrates the expansion of murine ASCs following their isolationusing similar protocols. Similar growth data has been obtained with ratASCs as shown in FIG. 1C).

These experiments demonstrate that ASCs are readily isolated and rapidlyexpanded ex vivo from relatively small amounts of adipose tissue, thuslaying the groundwork for using autologous ASCs in research and clinicalsettings. As discussed further below, neuroprotective studies suggestthat 30% of culture media replaced by the culture media from 10⁸ to 10⁹autologous cells exerts significant neuroprotective effects on differentneurodegenerative models.

B. Preparation of ASC-Conditioned Media

The ASCs were cultured in EGM2MV medium to confluence, and then switchedinto growth-factor free basal media (EBM-2, Clonetics) for conditioningin either normoxic or hypoxic conditions for 72 hours. Cell supernatantswere collected and subsequently assayed for cytokines using a RayBioarray VI and VII. FIG. 3 displays the findings of array VII, andindicate that in addition to the pluripotency of ASCs, the endocrine orparacrine potential of ASCs may have significant therapeutic relevance.Testing as shown that ASCs delivered to the CNS in the setting ofdegeneration as a result of ischemia due to stroke in this case may beable to protect neurons from death processes as well as enhanceangiogenesis by both differentiating into vascular phenotypes, and byrecruiting resident mature vascular endothelial cells to integrate intothe nascent vascular network. More importantly, in experiments directedto determining the overall biological effect of the ASC conditionedmedia, human microvascular endothelial cells were exposed to the mediaconditioned by ASCs incubated in (1) normoxic conditions, (2) hypoxicconditions, and (3) unconditioned media as a control. As shown in FIG.4, after 4 days, exposure to ASC-normoxic media resulted in an 80%increase in HMVEC number, while the ASC-hypoxic media resulted in a 160%increase in HMVEC number as compared to the control. These significantincreases confirm the potential of ASC conditioned media to promotegrowth or survival of other cells in their vicinity.

C. Isolation of Components in ASC Conditioned Media by Fractionation

Our studies illustrate that ASCs Conditioned Media during culture playsa critical role in protecting neurons from injurious stimuli. Definedprotein fractions (<10 k, 10-30 k, 30-50 k, and >50 k) from theconditioned media have been evaluated with respect to their ability toblock neuronal death in specific, well-defined in vitro models. Datashows that the passage number of the ASCs as well as variousenvironmental stimuli influence the level and composition of factorsecreted into the media and the resulting neuroprotective efficacy ofconditioned media.

Neuronal death is mediated by complementary neuronal death pathways indistinct neurodegenerative models, and may be limited in each model bydistinct trophic factor(s) by ASCs within the conditioned media.Neuroprotective factors the conditioned medium, such as VEGF and HGF,can be selected for by selectively altering the activity of presentfactors through the addition of inactivating antibodies, or conversely,by adding purified recombinant proteins to fractionated supernatants.

Evaluation of the Neuroprotective Capacity of Different Fractions ofMedia from ASCs in Different in vitro Neuronal Death Models.

Factors secreted into the media during culture of ASCs potently protectneurons from injury stimuli associated with specific neuronal deathpathways. Defined fractions of conditioned media from human, murine andrat ASCs, at different passages and subjected to hypoxia or normoxia,have been enriched by >50-fold in order to evaluate increase the potencyof neuroprotection and to enable detection of low abundance proteins byproteomic assays. Once the conditioned medium is enriched, it isoptionally fractionated by, for example, size exclusion and then can beconcentrated. Optionally, ASC Conditioned Media is simply concentratedand/or fractionated. In this manner, concentrations of the ASCConditioned Media can be manipulated.

According to another embodiment, ASCs derived from human, mouse or ratadipose tissue, as described above are evaluated by flow cytometry toconfirm that they are CD34+, CD90+, c-kit−, CD31−, CD45− and, in thecase of murine cells, Sca1+. The cells are then plated in DMEM/F12 with10% FBS with no additional growth factors added; and EGM/2MV (Cambrex)is used. The resulting ASC Conditioned Media is thereafter applied fortreatment or prevention of neural injury.

ASC Conditioned Media is enriched and size fractionated supernatants canbe further fractionated using a centrifugal filter device (10K, 30K, and50K Centriplus, Amicon, Mass.). Centriplus molecular weight filters canprovide an 100-fold sample enrichment and can easily be used to enrichsamples by 50 to 250-fold using a 10 kDa. Neuroprotective factors indifferent fractions of the supernatant are likely to be predominantlywithin the ranges of <10 kDa, 10-30 kDa, 30-50 kDa, and >50 kDa. It isknown that growth factors, for example, have a large size difference(such as GCSF 20 kDa BDNF 27 kDa EPO 34 kDa VEGF 45 kDa, and NGF 116kDa) based on size separation and will segregate in different molecularweight fractions. Optional steps include collecting and fractionatingsupernatants from fresh or passaged ASCs grown under normoxic or hypoxicconditions, and using these fractions for neuroprotection in LK/HK,glutamate, H2O2, ODG, 6OHDA, and MPP+ CGN models.

D. Delivery, Timing, and Dosing of ASC Conditioned Media

1. Delivery

Unlike cell therapies that inject stem cells at the point of injury, theprocess for treatment of injured nervous system cells, or cells prone toinjury or neurodegenerative diseases does not require localizedinjection. Rather, it will be appreciated that since no living cells,which may die if used systemically, are being utilized, that a widearray of delivery systems may be used to ensure that the ASC conditionedmedia, its fractions, concentrations, or distillations may be deliveredsystemically, via injection, intravenously, or otherwise. Optionally,the ASC conditioned media may be delivered locally at the site ofinjury. For example, the ASC Conditioned Media may be deliveredinterarterially, intravenously intraparenchemally, intrathecally, orinterperitonally.

2. Timing

In certain models, such as in the H-I model, that at 24 h followinginduction of hypoxia, the Blood Brain Barrier (“BBB”) is disrupted,allowing peptide penetration. Additionally, some growth factors, such asGCSF and IGF-1, can penetrate into the brain immediately after H-Itreatment. Identifying key factors for neuroprotection, initiallyconcentrated conditioned ASC Conditioned Media were used. 7 day old pupsunderwent will undergo H-I, followed by iv injections of 1-10 μl of250-fold concentrated rat ASC conditioned supernatant fractions or acocktail with defined growth factors at 2, 8 and 24 hours post surgery.As a control, animals were injected with the same amount of BME media.

The first few hours following H-I are believed to be the most criticalfor neuronal death resulting from direct effects of the insult.Secondary damage, triggered by inflammation, occurs after 48 hours.Given the prolonged period in which damage occurs, it may be beneficialto repeat dosing in order to effectively block neuronal death.Additionally, the ASC conditioned media or fraction thereof may havefunction to regenerate neurons derived from stem cells. Accordingly, theASC Conditioned Media or fraction thereof is optionally administered atan optimized concentrate at least once daily for at least one day, atleast 2 days, or at least 3 days following insult (such as H-I insult,onset of neurodegeneration, or surgery). Further optionally, due to theneural protection shown when ASC conditioned media or a fraction thereofis administered prior to insult, the ASC conditioned media may beoptionally administered at least one day, at least 2 days, or at least 3days prior to the insult or surgery.

3. Dosage

According to one aspect of the present application, a therapeutic doseof the ASC conditioned is delivered to an individual. In one embodiment,a defining characteristics of the ASC conditioned media arenaturally-derived factors secreted into the medium during fermentationof ASCs. This conditioned medium (CM) possesses the qualities of beingable to prevent damage to neurons due (a) ischemic events, (b) inductionof cell-death processes (apoptosis), (c) exocitotoxity, (d) oxidation,or (e) neuron-specific damaging agents. In vitro assays for each wouldbe (a) oxygen-glucose deprivation (OGD), (b) Low K model, (c) glutamateexocitoxicity, (d) hydrogen peroxide, and (e) 6-hydroxy-dopaminetoxicity of dopaminergic neurons at a therapeutic dose.

According to one embodiment, the ASC-CM is concentrated at least 50fold, at least 100-fold, at least 200 fold, or at least 1000-fold.Optionally, the concentrated ASC-CM is fractionated through a sizeexclusion resin or membrane to remove substances less than 5 kDa, lessthan 10 kDa, less than 20 kDa, less than 30 kDa, less than 40, kDa, orless than 50 kDa. The concentrated ASC-CM is then optionally stabilizedto protect degradation or loss of components. According to one exemplaryembodiment of dosing, 800 MICROLITERS/kg and up to 4000 MICROLITERS/kghave demonstrated efficacy in animal models when delivered as a singlebolus to the jugular vein, either before or after carotid arteryligation. However, according to alternative embodiments, dosing of about200 to 10,000 microliters per kg, about 600 to 2,000 microliters per kg,and about 1,000 to 1,200 microliters per kg may be delivered as a singlebolus as a therapeutic dose.

Turning to FIG. 19, according to another embodiment of the presentdisclosure, PC12 cells were cultured in DMEM containing 10% FBS for 3days, then starved in BME medium without FBS for 24 hours. Variouspercentages of the medium was exchanged for an equivalent volume of ASCconditioned media, as indicated on the figure above. The cells werecultured for 8 days in these media, which were replaced with freshlymade equivalent media every second day. The number of cells that formeda neuronal phenotypes were quantitated using a phase-contrastmicroscope. The results are expressed as the mean percentage ofneurite-bearing cells±sd, indicating that the ASC conditioned mediainduces differentiation to neurite-bearing cells.

III. Experimental Support of Efficacy Across Neuronal DegenerativeModels

It will be appreciated that treatment of neural tissue according tocertain embodiments disclosed in the present application were evaluatedagainst several in vitro neuronal degenerative models to demonstrate theeffectiveness of the treatment and composition with regard to multipleand varying types of damage that can induce neuronal death. These modelsare well established tools for the study of the CNS and peripheralnervous system related diseases, disorders and injuries. (Ni, 1997; Du,1997a; Du, 1997b; Dodel, 1998; Dodel, 1999; Du, 2001; Lin, 2001; Lin,2003). The use of these various models, which produce reasonable similesof prevalent human diseases, are particularly powerful tools for thestudy of the broad range of effectiveness of the ASC Conditioned Mediabecause each model is defined by distinct mechanisms involving variedpathways of degeneration. Furthermore, it is well known to thoseknowledgeable in the art that interference of the distinct mechanismsinvolved in cellular degeneration is limited to discrete factors and,furthermore, that individual factors that act on one mechanism have noeffect on others. Individual trophic factors can modify only discretedegenerative pathways or mechanisms. Therefore, a specific factor wouldbe expected to protect neurons from degenerative mechanisms involved inspecific neuronal death models, but would not provide any protection inmodels involving damaging agents that induce unrelated mechanism.Therefore, a single factor is unable to protect neurons from allneuronal death mechanisms. However, a mixture of factors, as is presentin ASC Conditioned Media, would provide the full complement of factors,acting individually or in combination, necessary to block alldegenerative mechanisms causing cell death. In FIG. 16 it is shown thatASC Conditioned Media possesses a factor (BDNF) that protects neuronsfrom glutamate-induced death. Neutralization of this factor greatly, butnot totally, reduces cell death in this model. Conversely, tests in anin the mechanistically distinct LK/HK death model demonstrated byneutralization of BDNF that this factor is not important for modifyingmechanisms leading to neuron death in this model. Therefore, BDNF as anindividual factor, as example, would not protect neurons from allmechanisms causing neuron death.

Detailed descriptions of the major neuronal death pathways, theinvolvement of each pathway in the models used in this study and therelevance of each model to human disorders is described in greaterdetail as follows below.

A. Mitogen-Activated Protein Kinase

Mitogen-activated protein (MAP) kinases are widely expressedserine-threonine kinases that mediate important regulatory signals incells. Three major groups of MAP kinases exist: the extracellularsignal-regulated (ERK) kinase family, the c-Jun NH₂-terminal kinase(JNK) family, and the p38 MAP kinase (p38) family. The members of thedifferent MAP kinase groups participate in the generation of variouscellular responses including gene transcription, induction of celldeath, maintenance of cell survival, malignant transformation, andregulation of cell-cycle progression (Widmann, 1999). The ERK-pathway isactivated in response to several cytokines and growth factors andprimarily mediates mitogenic and anti-apoptotic signals (Chang, 2001).There are three isoforms of JNK. At least one of the JNK₁₋₃ MAP kinasesis activated in response to stress and growth factors and similarlymediates signals that regulate apoptosis, cytokine production(inflammation), and cell-cycle progression (Davis, 2000). JNK signalinghas been shown to be involved in transient hypoxia-induced apoptosis indeveloping brain neurons (Chihab, 1998) and targeted deletion of JNK₃protected adult mice from brain injury after cerebral ischemia-hypoxia(Kuan, 2003). Additionally, blockade of JNK rescues neurons againstpotassium deprivation-induced CGN death (Xifro, 2005) andglutamate-induced neurotoxicity (Munemasa, 2005). p38 MAP kinase wasdiscovered as a major protein activated by LPS in macrophages and hasbeen characterized as the target for anti-inflammatory drugs thatinhibit IL-1 and TNF biosynthesis in monocytes (Lee, 1994; Han, 1994).Members of the p38 MAP kinase group are primarily activated by stressstimuli, but also during engagement of various cytokine receptors bytheir ligands (Lee, 1994; Lu, 1999; Rincon, 1998; Wysk, 1999). Thefunction of p38 kinases is required for the generation of variousactivities including regulation of apoptosis and cell cycle arrest,induction of cell differentiation, as well as cytokine production andinflammation (Dong, 2002). p38 MAP kinase also phosphorylates and/ormodulates the activity of a number of transcriptional factors involvedin cytokine responses including STAT1, IFN_(γ) regulatory factor-1, andNF-κB (Beyaert, 1996; Vanden Berghe, 1998). Recently, it has beenreported that inhibition of p38 MAP kinase significantly inhibitsNO-(Ghatan, 2000; Oh-hashi, 1999; Du, 2001), glutamate-(Kawasaki, 1997)and possibly hypoxia-ischemia-induced neuronal death (Hee, 2002).

Many of the genes responsible for apoptotic cell death, including thoseunderlying neuronal apoptosis, have now been identified and named ascaspases (Du, 1997a). Apoptotic cell death is often mediated by acaspase cascade. Although many stimuli exist, the final phases ofapoptosis are executed by a few common effector caspases. Mitochondriaappear to provide a link between the initiator caspases and thedownstream effector caspases. In non-neuronal cells, mitochondria havebeen shown to accelerate activation of caspases by releasingpro-apoptotic molecules, such as cytochrome c (Yang, 1997). MAP kinasessuch as JNK and p38 have been reported to regulate caspase 3-mediatedcell death (Harada, 1999; Cheng, 2001). However, it has also beenreported that c-Jun and p38 MAP kinases do not induce neuronal deaththrough the caspase-3 pathway (Sang, 2002; Roth, 2000). We haveidentified the involvement of caspase 3 in cytochrome c-mediated,glutamate-(Du, 1997a), MPP-(Du, 1997b), 6-hydroxdopamine-(Dodel, 1999),and potassium-deprivation-induced neurotoxicity (Ni, 1997).Additionally, it has been suggested that caspase 3 plays a role in therat H-I model (Turmel, 2001). Further, it has been documented thatcytochrome c and caspase 3 have more important function in the prematurebrain than the mature brain (Xu, 2004).

B. LK Induced CGN Apoptosis Model of Neuronal Cell Death

To induce apoptosis under the LK CGN model disclosed by Ni in 1997, CGNmaintained in BME with 25 mM potassium are switched to regular BME (5 mMpotassium) without serum and CGN (>50%), which induces apoptosis within24 h (Ni, 1997). This model was one of the first to be established, andis still widely used in studies of neuronal apoptosis in the primarycerebellar granule neuron (CGN) culture system, although its preciserelevance to the disease remains unclear (D'Mello, 1993; Dudek, 1997,Ni, 1997).

In the developing rodent cerebellum, granule neurons undergodevelopmentally regulated apoptosis peaking at the end of the first weekof postnatal life (Wood, 1993). Granule neurons cultured from rats ormice around this time of development undergo cell death in cultureunless they are provided with extrinsic survival factors. Maximalsurvival is produced by the combination of growth factors typicallyprovided by serum together with neuronal activity which is induced byhigh extracellular concentrations of potassium chloride that depolarizethe membrane and induce activation of voltage-sensitive calcium channels(D'Mello, 1997; Padmanabhan, 1999; Miller, 1996; Catterall, 2000). Thesignaling mechanisms by which growth factors and neuronal activitypromote the survival of CGN are beginning to be characterized. Proteinkinase cascades figure prominently in the control of neuronal survival.The ERK1/2-Rsk, phosphatidylinositol 3-kinase-Akt, and ERK5 proteinkinase signaling pathways play critical roles in mediating the survivalof CGN upon exposure to the neurotrophin brain-derived neurotrophicfactor (Bonni, 1999; Shalizi, 2003). The phosphatidylinositol3-kinase-Akt signaling pathway plays a central role in mediatinginsulin-like growth factor 1-mediated neuronal survival (Brunet, 2001).Removal of survival factors promotes neuronal apoptosis in part becauseof inactivity of pro-survival protein kinases. However, deprivation ofsurvival factors also leads to stimulation of other protein kinases thatimpart an apoptotic signal in neurons. These protein kinases includeJNK, p38, Cdc2, and GSK3 (Harada, 1999, Estus, 1994; Xia, 1995; Watson,1998; Yang, 1997; Donovan, 2002; Konishi, 2002; Konishi, 2003; Mora,2001).

C. Glutamate Induced Model of Neuronal Cell Death

According to testing protocol for the glutamate induced neuronal deathmodel, neuronal apoptosis or necrosis is induced in CGN with 30-100 μMglutamate or cortical neurons (CN) with 100 μM of NMDA. Glutamate is anexcitatory neurotransmitter used throughout the central nervous systemand is associated with various brain functions, such as synapticplasticity, learning, and long-term potentiation (Collingridge, 1989).Its physiological and pathological effects in the CNS are mediatedmainly via two types of ionotropic glutamate receptors, the NMDAreceptor and the non-NMDA receptor. When present in excessiveconcentrations glutamate has the potential to induce serious damage andeven death of neurons (Lucas, 1957), with N-methyl-D-aspartate (NMDA)receptors located on neuronal cell bodies playing a major role in thisexcitotoxicity (Rothman, 1987). NMDA receptor activation allows aninflux of calcium through both glutamate-activated cationic channels(NMDA) and voltage-gated Ca²⁺ channels activated by a prolongeddepolarization (Choi, 1987; Coulter, 1992; Olney, 1971). Althoughincreases in intracellular calcium concentrations are a necessarycomponent of many normal signal transduction pathways, excessive andprolonged rises of Ca²⁺ can lead to mitochondrial membrane dysfunctionand cell death (Farber, 1981; Sombati, 1991), induced in part byCa²⁺-mediated excitotoxicity (Wahlestedt, 1993) and/or failure toregulate cell volume (Pasantes-Morales, 2000). Cell death associatedwith glutamate neurotoxicity has been suggested to contribute to thedevastating effects of a number of serious medical conditions includingstroke, persistent seizures of status epilepticus, and neurodegenerativedisorders such as Alzheimer's disease, amyotrophic lateral sclerosis,multiple sclerosis, spinal cord injury and Huntington's disease (Choi,1988; Choi, 1990; Kandel, 1991).

It has been reported that reactive oxygen species (ROS) are generated byactivation of the glutamate receptor (Campisi, 2004). Additionally, MAPkinases including JNK and p38 are also implicated in glutamate-inducedneuronal apoptosis (Xia, 1995; Chen, 2003). Furthermore, caspase 3activation appears to play an important role in glutamate neurotoxicity(Du, 1997).

D. Hydrogen Peroxide Induced Model of Neuronal Cell Death

According to the hydrogen peroxide induced death model, we treated CGNwith 10 μM of H₂O₂ to induced neuronal death (Lin, 2003). It has beensuggested that hydrogen peroxide leads to apoptotic neuronal death byinvolving pro-apoptotic molecules (Wei, 2000), like initiator caspases(See, 2001). Superoxide anions seem to be responsible for the apoptoticcell death of trophic factor-deprived sympathetic neurons (Greenlund,1995a; b), glutamate-treated cerebellar neurons (Ishikawa, 1999; Satoh,1998; and Patel, 1996), and hippocampal neurons incubated with xanthineoxidase (Guo, 1999; Ishikawa, 1999). Singlet oxygen has also beeninvolved in apoptotic death in nonneuronal cells mediated by Bid andsome members of the MAPK family (Zhuang, 1998). In addition, singletoxygen has been related to the alterations in the mitochondrialpermeability transition pore that occur in several apoptotic deathmodels (Salet, 1997; Moreno, 2001). ROS contributes to the production ofperoxynitrites and could also have relevance in induction of apoptoticcell death (Virag, 1998).

E. Nitric Oxide Induced Model of Neuronal Cell Death

Treatment of CGN with 50 μM of sodium nitroprusside (SNP, a NO donor)induces neuronal death (Lin, 2001). Nitric oxide (NO) generated fromneuronal nitric oxide synthase (nNOS) and inflammatory inducible isoformof nitric oxide synthase (iNOS) inhibits the mitochondrial respiratorychain in vitro (Clementi, 1998), stimulates neurotransmitter releasefrom synaptosomes (Meffert, 1994) and can cause autocrine excitotoxicityin neuronal cultures (Leist, 1997). NO plays a critical role inneurodegenerative diseases and cerebral ischemia. It has been suggestedthat excessive production of NO causes these diseases by destroyingneurons. The mechanisms proposed for NO-mediated neurotoxicity includeinactivation of the mitochondrial respiratory chain (Heales, 1994),S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (McDonaldand J. Moss, 1993), inhibition of cis-aconitase (Drapier, 1993),activation of poly (ADP-ribose) synthase, and DNA damage (Zhang, 1994),most of which can be mediated by the formation of nitrosocompounds bycellular components. Additionally, p38 MAP kinase and cGMP-dependentprotein kinase (PKG) have been implicated in NO— induced neuronalapoptosis (Ghatan, 2000; Lin, 2001; Bonthius, 2004).

F. 6-Hydroxy Dopamine Neuronal Model of Neuronal Cell Death

Treatment of CGN or dopaminergic neurons (DA) with 6-hydroxydopamine(“6-OHDA”) induces neuronal death (Dodel, 1999). 6-OHDA is a neurotoxinthat is specific for catecholamine/dopaminergic neurons (DN) in both thecentral and peripheral nervous systems. This neurotoxin has been widelyused for the Parkinson disease (PD) research. It has been hypothesizedthat 6-OHDA induces neuronal death possibly via uncoupling mitochondrialoxidative phosphorylation resulting in energy deprivation (Glinka,1996). Alternatively, 6-OHDA-induced neurotoxicity has been associatedwith its rapid auto-oxidation at neutral pH, thus producing hydrogenperoxide, hydroxyl and superoxide radicals (Kumar, 1995;Tiffany-Castiglinoi, 1982). Quinones formed during the auto-oxidation of6-OHDA may undergo covalent binding with nucleophilic groups ofmacromolecules such as —SH, —NH₂, —OH, possibly further enhancing6-OHDA-induced neurotoxicity (Izumi, 2005). Furthermore, peroxynitrite(ONOO⁻), which is a potent oxidant formed during the nearlyinstantaneous reaction of nitric oxide with superoxide anion, has alsobeen found to be involved in 6-OHDA-induced neurochemical effects(Ferger, 2001) and neurotoxicity (Mihm, 2001). Peroxynitrite-mediatedprotein nitration has been well documented in neurodegenerativedisorders including Parkinson's disease (Beckman, 1993; Good, 1996,1998). We used CGN since CGN undergo cell death as do dopaminergicneurons when exposed to 6-OHDA and MPP⁺ (Dodel, 1999; Du, 1997a).Importantly, neuronal death pathways have been better characterized inCGN since this system provides a pure neuronal culture (Dodel, 1999).

G. MPP+ Induced Neuronal Model of Neuronal Cell Death

Treatment of CGN or dopaminergic neurons (DA) with MPP⁺ induces neuronaldeath (Du, 2001). MPTP/MPP⁺-induced neurodegeneration of DAN and CGN iswidely used to investigate and characterize the pathogenesis of PD (Du2001). MPP⁺ is incorporated into cells via the dopamine transporters andthe main targets of MPP⁺ are the mitochondria, where it inhibits ComplexI in the respiratory chain and abolishes oxidative phosphorylation(Tipton, 1993). Although the cerebellum has not been extensively studiedas a target for MPP⁺ neurotoxicity, CGCs are quite sensitive to thetoxic effects of MPP⁺ in vitro (Du, 1997a; Gonzalez-Polo, 2003). Theneurotoxic action of this compound is known that MPP⁺ binds to complex-Iof the mitochondrial respiratory chain, causing the inhibition ofNAD-linked mitochondrial respiration (Javitch, 1985), the increase inthe generation of reactive oxygen species (Akaneya, 1995) and caspase-3activation (Du, 1997a). It has been also suggested the regulatoryeffects of MPP⁺ on the N-methyl-D-aspartate (NMDA)-receptor, inducingthe Ca²⁺ entry into the cell (Robinson and Coyle, 1987).

H. Hypoxia-Ischemia Neuronal Model of Neuronal Cell Death

Hypoxic-ischemic (H-I) encephalopathy during the prenatal and perinatalperiod is a major cause of damage to the fetal and neonatal brainresulting in considerable morbidity and mortality (Wei, 2004). However,currently, there is no effective treatment to prevent the consequencesof neonatal H-I in humans. Both rat and mouse in vitro and in vivomodels of neonatal H-I have been established for mechanistic study.Hypoxic-ischemic insults can trigger both apoptosis (delayed programmedcell death) and necrosis. It has been reported that young neurons die ofnecrosis and delayed apoptosis (Northington, 2001), whereas adultneurons usually die of necrosis only (Walton, 1999). This difference ismainly due to the upregulation of NMDA receptors and increased caspase-3activity in the young brain and these two factors make young neuronsparticularly vulnerable to H-I injury (Johnston, 2002). Mitochondriaappear to play an essential role in determining the fate of cellssubjected to hypoxia-ischemia (Gilland, 1998). Disrupted mitochondrialfunction during H-I can lead to cytochrome c protein release and triggeran activation of caspase 3/other caspase-related apoptotic pathways(Cheng, 1998). Additionally, Calpain and neuroinflammation may also beinvolved in H-I-induced neuronal injury (Arvin, 2002, Wei, 2004). Theprominence of both apoptosis and necrosis in neurodegeneration after H-Iin the immature brain suggests that it will be important to betterunderstand the roles and relationships of these processes to developeffective neuroprotective strategies.

I. Oxygen and Glucose Deprivation Neuronal Death Model

The in vitro oxygen and glucose deprivation (OGD) model highlycorrelates to mechanisms of action in the in vivo H-I model. We culturecortical (CN) or hippocampal (HN) neurons from 1-d pups and after 7-dsubject neurons to two hours of hypoxia in media without glucose (seemethod for details). This model can be used for mechanistic study of invivo hypoxia-ischemia-induced neuronal injury.

In summary, the table below lists some of the major neuronal deathpathways that are involved in the above-mentioned models.

TABLE 1 Models LK Glu H₂O₂ 6OHDA MPP NO OGD Cell type CGN CGN, CGN CGN,CGN, CGN CN, HN CN DN DN Necrosis (N) or A N and A N and A N and A N andA N and A N and A apoptosis (A) Caspase 3 weak strong weak weak strongweak modest JNK modest modest weak weak modest weak modest p38 modestmodest weak weak weak strong modest Transcription/translation strongweak weak weak weak weak weak blocker Antioxidant weak* weak* StrongSome Some Some Strong modest Strong Strong for some Physiology relevanceApoptosis Ischemia All PD PD PD and Hypoxia model and neurodegenerativeinflammation- and others disorders related ischemia neuronal death *Someantioxidants may have neuroprotective functions through non-antioxidantfunctions.

J. Assessment of Neuroprotective Effects Using Neurodegenerative Models.

The in vitro and in vivo neuronal death models were used to quantify theneuroprotective effect exerted by ASCs conditioned media. These methodswere further used to show the efficacy of using various fractions andcomponent of ASC conditioned media to produce significantneuroprotective effects on different neuronal death pathways. Thesetechniques can also be used in vivo neonatal H-I model to examinewhether the conditioned medium or factors identified therein can besystemically delivered to exert neuroprotection in vivo.

IV. Results of Testing ASC-CM Against Use of Neurodegenerative Models

When the following neurodegenerative models were used by incorporatingASC-CM into in vitro cultures, or according to protocol set forthherein, the following results were noted.

A. ASC Conditioned Media Protects CGN Against Glutamate-Induced NeuronalDeath.

Glutamate induces both neuronal necrosis and apoptosis and this in vitromodel has been widely used for research of stroke, Parkinson's disease,and Alzheimer's disease (Du, 1997). In order to understand thephysiological relevance of ASC Conditioned Media, in the glutamate modelwas used to test neural cells cultured on media containing ASCConditioned Media. As shown in FIG. 6, the conditioned medium from ASCssignificantly protected neurons against glutamate neurotoxicity.Furthermore, FIG. 7 demonstrates that ASCs which had not been previouslycultured (“fresh”) produce an ASC conditioned Media that possesses ahigher potency in neuroprotection than ASCs that are not fresh. Further,FIG. 8 indicates that ASC Conditioned Media that still retains theprotective/regenerative characteristics fractionate by size exclusionchromatography at apparent molecular weights in excess of 10 kDa.

B. Human ASC-Conditioned Media Protects CGN Against Glutamate-InducedRat Neuronal Death.

Human ASC Conditioned Media was tested in a rat glutamate toxicitymodel. The results indicate that human ASC Conditioned Media, like thatof rat, significantly attenuated glutamate neurotoxicity in rat CGN,suggesting that ASC Conditioned Media induce activity in rat cells.Thus, the rodent model can be useful for assaying the neuroprotectiveproperties of human ASCs.

C. ASC Conditioned Media Protects CGN Against H₂O₂-Induced NeuronalDeath.

The H₂O₂-induced neuronal death model shows the role of free radicals inneurodegeneration. Free radicals have been implicated in almost alltypes of neurodegenerative processes. Test results shown in FIG. 12demonstrate that ASC Conditioned Media exerted potent anti-oxidantactivity, thereby protecting CGN from oxidative damage and death.

D. ASC Conditioned Media Protects Against OGD-Induced Cortical NeuronalDeath.

The oxygen and glucose deprivation (OGD) model highly correlates tomechanisms in action in the in vivo H-I model. The protective effect ofASC Conditioned Media was tested when added to primary mouse corticalneurons from 1-d old pups. The cultured neurons were placed in Hanksbuffer without glucose and incubated for 2 hours in a hypoxic chamber(Form a Scientific) that was preset at 37° C. and 1% O₂. Neurons werethen switched back to serum-free DMEM medium in the presence or absenceof ASC Conditioned Media. 24 hours later, neurons were assayed by an LDHkit. As a control, neurons without OGD treatments were also switchedinto serum-free DMEM media in the presence or absence of ASC ConditionedMedia to eliminate LDH effects from ASC Conditioned Media. Test resultsshown in FIG. 13 indicate that ASC Conditioned Media markedly protectsneurons against OGD-induced neuronal injury.

E. 250× Enriched ASC Conditioned Media Protects Neurons AgainstH-I-Induced Hippocampal Neuronal Death In Vivo.

To investigate ASC Conditioned Media function in H-I-induced neuronaldeath in vivo, 7-d old Sprague Dawley rat pups were anesthetized with2.5% halothane and the left carotid artery was permanently ligated.Hypoxic exposure was then achieved by placing pups in a 2.0-L airtightplastic chamber submerged in a 37.0° C. water bath and flushed for 2 hwith a humidified mixture of 7% oxygen and 93% nitrogen. Pups were thenreturned to their dams until sacrifice. Pups (2 per group) receivedIntravenous (i.v.) injections of 10 μl of 250-fold concentrated rat ASCConditioned Media 24 h after the hypoxic insult.

The time period between H-I induction and ASC Conditioned Mediainjection was chosen because maximal disruption of the blood-brainbarrier occurs at this time, allowing maximal penetration of largepolypeptides into brain tissues (Ikeda, 1999; McLean 2004. Seven daysfollowing H-I injury, the brains were histologically analyzed toquantify the amount of damage to the hippocampus. In the hippocampus,H-I injury resulted in approximately 27% tissue loss when mice wereexposed to hypoxia for 2 hours, as compared to non-injured controls.Conversely, FIG. 12 shows that mice treated with ASC Conditioned Mediashowed almost completely blocked brain damage.

As discussed above, ASC Conditioned Media effectively blocks neuronaldeath in models that involve different molecular mechanisms. Thesemechanisms include at least one of the following three pathways: JNK,p38, and caspase 3. These three pathways have been widely investigatedand it is known that in addition to interacting each other, thesepathways may also induce neuronal death independently (see Table I).Using these models, it is possible to determine if ASC-conditioned Media(ACASC Conditioned Media) inhibits injury stimuli-induced activation byphosphorylation of JNK and p38 and cleavage of caspase 3 in those modelswhere they are actively involved (Table 1). According to our embodiment,a ASC Conditioned Media prepared as described protects neurons fromneuronal death in these models via inhibition of ENK, JNK, p38, and/orcaspase 3 activation.

The adipose tissue is minced (mouse and rat) then digested inCollagenase Type I solution (Worthington Biochemical, Lakewood, N.J.)under gentle agitation for 1 hour at 37° C., filtered with 500 μm and250 μm Nitex filters, and centrifuged at 200 g for 5 minutes to separatethe stromal cell fraction (pellet) from adipocytes. The ASC fraction istreated with red blood cell lysis buffer for 5 min at 37° C., thencentrifuged at 300 g for 5 minutes. The supernatant is discarded and thecell pellet resuspended in the appropriate medium.

F. ASC Conditioned Media Protects Against OGD-Induced Cortical NeuronalDeath.

ASC-CM protects neurons against 6-hydroxydopamine (6-OHDA)-mediateddeath, as shown in FIG. 18. The ASC Conditioned Medium (ASC-CM) wascollected and subsequently added to the cultured rat cerebellar granuleneurons (CGN). Neuronal viability was quantified by either countingfluorescein positive neurons or staining living neurons with MTT. Sinceneurotoxicity induced by 6-OHDA was believed to be due, at least inpart, to the production of reactive oxygen species (ROS). Alsoinvestigated were the levels of free radical generation in our model byusing dihydroethidium (DHE) and dihydrorhodamine 123 (DHR). As shown inFIG. 18, exposure of CGN to 50 mM 6-OHDA resulted in significantincreases in free radical production and CGN neuronal death.

G. ASC-CM Preserves the Cognitive Function of Rats FollowingHypoxia-Ischemia injury.

The ability of ASC-CM to provide long-term protection followinghypoxia-Ischemia (HI) injury was determined as follows. HI injury wasinduced in 7 day old rat pups as described above. ASC-CM wasadministered at the time of surgery (pre-treatment) or 24 hours after HIinjury (post treatment). Controls were uninjured rats (positive control)of the same age and rats receiving an equivalent volume of carrier(negative control). After 7 weeks the cognitive function of all rats wasdetermined using the Morris Water Maze test.

The test system consisted of a swimming pool containing a number ofvisual cues to facilitate orientation, including counters and decals. A168 cm diameter, 41 cm high tank was filled to a depth of 30 cm with 15°C. water. A round transparent plastic platform, 1 cm in diameter, wasplaced in the pool so that the top of the platform was located 1 cmbelow the surface of the water, where it was not visible to a viewer onthe surface of the water. For the visible platform test, a flag wasplaced on the platform. After performing the visible platform test, theflag was removed, making the platform not visible from the surface ofthe water (invisible test). Animals were individually placed at the samelocation in the water to begin the test. The time taken for the rats toreach the platform by swimming was recorded. Each animal was tested 3times with 15 second intervals between repeats. Data are presented asmean ±SEM. The results were compared using a paired Student's t-Test

As shown on FIG. 17, for the visible platform test the time taken by therats to first swim to the platform and then crawl out of the water ontothe platform was much shorter for both ASC-pretreatment (n=3) andASC-posttreatment (n=4) groups than for the control BME-treatment group(n=5) (**P<0.01). Similarly, ASC-CM treated rats performed better thanBME control-treated rats in the invisible platform test (*P<0.05) (FIG.2). These results demonstrate that ASC-CM treated rats have a higherlevel of cognitive function than control treated animals; thus,providing further evidence that ASC-CM provides protection againstneurodegeneration.

V. Methods

A. Preparation of Mouse and Rat CGN Neuronal Cultures and AnalysisMethod.

CGN is prepared from 8-day-old rat or mouse pups as previously described(Du, 1997 and 2001). Preliminary data showed that mouse CGN behavessimilarly to rat CGN. Briefly, freshly dissected cerebella isdissociated and the cells seeded at a density of 1.2 to 1.5×10⁶ cells/mlon poly-L-lysine coated dishes in basal medium Eagle (Invirogen)supplemented with 10% FBS (Invirogen), 25 mM KCl, and gentamicin (0.1mg/ml, Invitrogen). Cytosine arabinoside (10 μM, Sigma) is added to theculture medium 24 h after initial plating. All experiments utilizeneurons after 7-8 days in vitro (DIV). The LK, glutamate, H2O2, MPP+,6OHDA treatments follow methods that were previously described (Ni,1997; Du, 1997a; Lin, 2003; Du, 1997b; Dodel, 1999). Viable neurons arequantified by counting fluorescein (green) positive cells which resultfrom the de-esterification of fluorescein diacetate (FDA, Sigma) byliving cells. Briefly, cultures are incubated with FDA (10 μg/ml) for 5min, examined and photographed using UV light microscopy and the numberof neurons from representative low power fields are counted aspreviously described (Du, 1997). Propidium iodide (PI, Sigma), whichinteracts with nuclear DNA producing a red fluorescence, is used toidentify dead neurons. For PI staining, cultures are incubated with PI(5 μg/ml), examined and photographed using UV light microscopy aspreviously described (Du, 1997a).

B. Cultured Mesencephalic Neurons

Primary cultures of rostral mesencephalic tegmentum (RMT) dissected fromE15 rat or E12 mouse embryos (Harlan) are performed using a modifiedmethod as previously described (Dodel, 1998). Preliminary studies showthat mouse MDN behaves similarly to rat MDN. Briefly, RMT is dissociatedusing trypsin and DNase (Sigma) and the cells are be suspended inDulbeccos Modified Eagle Medium (Invitrogen) supplemented with Ham F12nutrient mixture (1:1; Invitrogen), glucose, 1% penicillin-streptomycin(Invitrogen) and 10% fetal bovine serum (Invitrogen). The cells areplated onto poly-L-lysine (10 μg/ml; Sigma) precoated 10 mm coverslipsin 24-well plates at a density of 10⁵ cells/cm² and incubated for 72 hr.Following 24 h the medium is supplemented with 10 μM cytosinearabinoside (Sigma) to inhibit glial cell proliferation. Neuronalcultures are used for experiments 7 days after preparation. Dopaminergic(DA) neurons in primary cultures are visualized byTH-immunohistochemistry using a primary monoclonal antibody against ratTH (Incstar) following by an anti-mouse IgG Cy3 conjugate (Sigma)(Dodel, 1998), and the number of TH-immunoreactive neurons is assessedusing a Leitz inverted microscope (×200). Values are usually expressedas a % of control cultures for each experiment and the data aredisplayed as the mean ±standard error of duplicate experiments, whichare repeated about four times. The cell counts are statisticallyevaluated using analysis of variance.

Neurotoxicity is also examined by using methods of TUNEL (APOPTAG,ONCOR) and LDH (Roche) following manufacturers' instructions (Dodel,1998).

C. Primary Neonatal Cortical Neuronal Culture

Cortices are collected from newborn rat or mice pups and minced. Analiquot of ice-cold PBS is added into the minced tissues, which are thencentrifuged at 1000 rpm at 4° C. and the supernatants are discarded. Analiquot of 0.25% trypsin is added and incubated at 37° C. for 15 min toproduce a single cell suspension (shaken once every 5 min). Theprecipitates are discarded and the supernatants are centrifuged again at1000 rpm at 4° C. for 5 min. The cell pellets are diluted to anappropriate concentration with Neurobasal in 2% B27 (Invitrogen,Carlsbad, Calif., U.S.A.) and plated into poly-d-lysine-coated dishes(BD Biosciences, Franklin Lakes, N.J., U.S.A.). Usually, the cells areused between 4-6 days after plating. Before each treatment, cells arerinsed and then incubated in serum-free Dulbecco's modified Eagle'smedium (DMEM) with or without high glucose (Invitrogen). All experimentsare conducted under serum-free conditions. To induce OGD, neurons areplaced in a hypoxic chamber (Form a Scientific) which is preset at 37°C. and 1% O₂. Neurons are incubated with serum-free DMEM mediacontaining no glucose. Control neurons are incubated in the regularincubation chamber (37° C. and 21% O₂) in DEME containing high glucose.Four hours later, neurons are placed back to regular CO₂ incubator foranother 20 h and then assayed using a LDH kit. For NMDA toxicity study,the neuronal culture is supplemented with 100 mM for 24 h and assayedusing a LDH kit.

D. MTT Assay.

Cell viability assays are performed in accordance with the protocolprovided by R & D Systems (Minneapolis, Minn., U.S.A.). Briefly,cortical neurons from newborn rats are cultured in flat-bottomed,poly-d-lysine-coated, 96-well tissue culture plates (BioCoat, BDBiosciences). After each treatment, 100 μl of media is removed for theLDH assay and MTT is added to the cultures at 37° C. for 2.5 h. DMSO isthen added to the cells. Cells are held for another 3 h at 37° C. in thedark since MTT is reduced by metabolically active cells into insolublepurple formazan dye crystals that are soluble in the DMSO. Theabsorption is read by a plate reader at 570 nm using a referencewavelength of 650 nm.

E. LDH Assay.

About 100 μl of the culture media is monitored for the release oflactate dehydrogenase (LDH) to measure cell death, using a LDH kit fromRoche, Indianapolis by following the manufacturer's instructions (Du,1998). Each experiment is performed in triplicate; the data from arepresentative experiment carried out three times with similar results.The data is expressed as the mean OD±SD.

F. TUNEL Assay.

DNA strand breaks are detected using terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end-labeling (TUNEL)according to the manufacturer's procedure (APOPTAG, ONCOR). Briefly,cultures are fixed for 30 min with 1% paraformaldehyde and then washedwith PBS. 200 μl of Equilibration buffer is added to each well, followedby addition of 120 μl/well of working strength TdT cells are, thenincubated for 30 min at 37° C. After adding 1 ml of working strengthstop/wash buffer twice, 100 μl of working strength antibody solution(anti-digoxigenin-fluorescein) is added the mixture is held for 1 hr.The cells are visualized under phase-contrast microscopy. Apoptoticcells are discriminated morphologically by the presence of condensed,bright green nuclei in neurons.

G. Western Blot Analysis.

Western blot analysis of ERK, JNK, p38, and active caspase is performedas previously described (Wei, 2004, Wei, 2005). Detection of caspaseactivity is also performed as described in 1997 (Du, 1997a,b). Neuronalextracts are prepared by lysing CGN at 1.5 and 3 h (for JNK and p38), 0,3 and 6 h (ERK), and 20 h (for caspase 3) following insult treatments.

H. Proteomic Profiling of Neuroprotective Factors in Fractions fromASC-Conditioned Media.

Neuroprotective factors secreted by ASCs, are characterized usingantibody arrays to identify specific factors present in fractions ofconditioned medium. This information is used to assess the contributionof each factor to neuroprotective activity. An antibody array is usedfor initial characterization over other proteomics analyses because thismethod is sensitive (can detect pg level) and can directly measure theprotein in media. In contrast, nucleic acid microarrays (SuperArray,Affymetrix, Agilent) can only detect mRNA changes and may not provideaccurate data on protein production and secretion in to the media. Othermethods for detecting proteins are much more powerful and can be used.Our data demonstrates that conditioned media from human ASCs potentlyprotects rat neurons, suggesting that cross-species analyses andprotection occurs.

H. Proteomic Profiling of Growth Factors/Cytokines Present inConditioned Media.

Active fractions from human and mouse ASC antibody can be identifiedusing array membranes provided by RayBiotech. Detailed methodology isdescribed in the company protocol; it is similar to Western blotprotocols. In brief:

Step 1. Incubate the array membrane with 250-fold enriched ASCssupernatants.

Step 2. Incubate the factor-bound membrane with a cocktail ofbiotin-labeled antibodies.

Step 3. Incubate the array membrane with HRP-conjugated streptavidin.

Step 4. chemoluminescent detection.

The presence of any proteins detected by the array analyses can beconfirmed in rat or murine (if not proved to be negative through probingthe mouse array) conditioned media using antibodies or RT-PCR analyses,and the identified proteins can be manufactured using CDNA or othermethods.

Because of the larger array of human antibodies, and conditionsavailable it is more effective to probe for human proteins than it is toprobe for specific proteins in rats, mice and other animals. However,mouse and rat ASCs may have unique properties that are not conservedacross species. We do not believe that this is the case since our datademonstrate that human ASC conditioned media is a potent protector ofrat neurons. However, the number of proteins detected by the arrays isstill a fraction of the total number of proteins present in the media.It is possible that important factors in the conditioned media could goundetected. Accordingly, it may be necessary to use methods other thanantibody detection to screen for factors in ACASC Conditioned Mediaproduced by rat or mice cells. One such method is to use oligonucleotidearray to identify components produced by ASCs. Commercially availableoligonucleotide arrays include SuperArray, Affymetrix, or Agilent. Theother arrays can be used to probe for factors that do not react withantibody assay. This technique provides information in the absence ofantibodies and can be used directly with mouse and rat cells. Ifnecessary, these two methodologies can be combined to overcome theseinherit deficiencies of each separate method.

All references, patents, patent applications and the like cited hereinand not otherwise specifically incorporated by references in theirentirety, are hereby incorporated by references in their entirety as ifeach were separately incorporated by reference in their entirety.

An abstract is included to aid in searching the contents of theapplication it is not intended to be read as explaining, summarizing orotherwise characterizing or limiting the disclosure in any way.

The present disclosure contemplates modifications as would occur tothose skilled in the art. It is also contemplated that processesembodied in the present disclosure can be altered, duplicated, combined,or added to other processes as would occur to those skilled in the artwithout departing from the spirit of the present disclosure.

Further, any theory of operation, proof, or finding stated herein ismeant to further enhance understanding of the present disclosure and isnot intended to make the scope of the present disclosure dependent uponsuch theory, proof, or finding.

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, the same is considered to beillustrative and not restrictive in character, it is understood thatonly the preferred embodiments have been shown and described and thatall changes and modifications that come within the spirit of thedisclosure are desired to be protected.

1. A method of producing a stem cell conditioned media, comprising thesteps of: a. providing at least one human adipose stem cell; b.providing at least one medium for culturing the stem cell; c. culturingthe at least one human adipose stem cell in the at least one medium; d.switching the medium to a growth-factor free basal media forconditioning the at least one human adipose stem cell in either normoxicor hypoxic conditions for 72 hours; and e. collecting cell supernatantseffective to treat neural insults.
 2. The method of claim 1, wherein theat least one medium is EGM2MV, and the human adipose stein cells arecultured to confluence prior to switching the medium to a growth-factorfree basal media.
 3. The method of claim 2, wherein the human adiposestem cell has a surface marker profile of lin-/CD45−/c-kit-/CD90+. 4.The method of claim 3, wherein the resultant supernatant is concentratedat least 50-fold.
 5. The method of claim 4, wherein the resultantsupernatant is fractioned to remove substances less than 10 kDa.
 6. Themethod of claim 4, wherein the resultant supernatant is fractioned toremove substances less than 30 kDa.
 7. The method of claim 3, whereinthe adipose stem cells display the surface marker profile CD34+positive.
 8. The method of claim 2, wherein the neural insults to betreated by the cell supernatants are selected from the group consistingof hypoxia-ischemia, encephalopathy, stroke neurodegenerative disorders,and chronic or acute injuries in nervous system.
 9. The method of claim2, wherein the adipose stem cells are obtained from lipoaspirates. 10.The method of claim 2, wherein the human adipose stem cell is harvestedfrom an individual to be treated for a neural insult.
 11. A method ofproducing a stem cell conditioned media, comprising the steps of: a.providing at least one human or rat adipose stem cell harvested from anindividual to be treated for a neural insult, the at least one stem cellcomprising an adipose stem cell harvested from a lipoaspirate; b.providing at least one medium for culturing the stem cell; c. culturingthe at least one human or rat adipose stem cell in the at least onemedium; d. switching the medium to a growth-factor free basal media forconditioning the at least one human or rat adipose stem cell in eithernormoxic or hypoxic conditions for 72 hours; and e. collecting cellsupernatants effective to treat neural insults and introducing the cellsupernatants to the individual in a therapeutic dose.